High Resolution Proteome of Lipid Droplets Isolated from the Pennate Diatom Phaeodactylum tricornutum (Bacillariophyceae) Strain pt4 provides mechanistic insights into complex intracellular coordination during nitrogen deprivation

Abstract

Lipid droplets (LDs) are an organelle conserved amongst all eukaryotes, consisting of a neutral lipid core surrounded by a polar lipid monolayer. Many species of microalgae accumulate LDs in response to stress conditions, such as nitrogen starvation. Here, we report the isolation and proteomic profiling of LD proteins from the model oleaginous pennate diatom Phaeodactylum tricornutum, strain Pt4 (UTEX 646). We also provide a quantitative description of LD morphological ontogeny, and fatty acid content. Novel cell disruption and LD isolation methods, combined with suspension‐trapping and nanoflow liquid chromatography coupled to high resolution mass spectrometry, yielded an unprecedented number of LD proteins. Predictive annotation of the LD proteome suggests a broad assemblage of proteins with diverse functions, including lipid metabolism and vesicle trafficking, as well as ribosomal and proteasomal machinery. These proteins provide mechanistic insights into LD processes, and evidence for interactions between LDs and other organelles. We identify for the first time several key steps in diatom LD‐associated triacylglycerol biosynthesis. Bioinformatic analyses of the LD proteome suggests multiple protein targeting mechanisms, including amphipathic helices, post‐translational modifications, and translocation machinery. This work corroborates recent findings from other strains of P. tricornutum, other diatoms, and other eukaryotic organisms, suggesting that the fundamental proteins orchestrating LDs are conserved, and represent an ancient component of the eukaryotic endomembrane system. We postulate a comprehensive model of nitrogen starvation‐induced diatom LDs on a molecular scale, and provide a wealth of candidates for metabolic engineering, with the potential to eventually customize LD contents.

Lipid Droplets in Unicellular Photosynthetic Stramenopiles

“…In the study performed by Lupette et al (2019) on mature LDs (7 days of nitrogen starvation), StLDP and PtLDP1 were both recorded in the LD proteome, although no major LD protein was pointed out. The presence of StLDP was further confirmed in a more recent analysis of LD proteome in the Pt4 ecotype of P. tricornutum (Leyland et al, 2020b).…”

“…SLC25A4 (an adenine nucleotide translocator) is located in the inner mitochondrial envelope membrane, supporting a possible connection of the LD with the mitochondrion (Lupette et al, 2019). Some of these protein components were confirmed in a more recent proteomic study of LD purified from the Pt4 ecotype of P. tricornutum (Leyland et al, 2020b).…”

“…On the other hand, the emergence of P2 and P3 population correlates with a decrease in polar glycerolipids, ventilating the hypothesis that most of their TAG content may derive from membrane lipid recycling. It must be noticed that the fine sequence of biogenesis of LD subpopulation deciphered in the Pt1 ecotype of P. tricornutum (Jaussaud et al, 2020) was not detected in the Pt4 ecotype (Leyland et al, 2020b). Further studies are needed to disentangle the origin, composition, and functions of the different populations of LD.…”

“…Several proteomics analyses have been performed in different stramenopiles, e.g., Nannochloropsis oceanica (Vieler et al, 2012a), Fistulifera sp. (Nojima et al, 2013;Nonoyama et al, 2019), and P. tricornutum (Yoneda et al, 2016;Wang et al, 2017;Lupette et al, 2019;Leyland et al, 2020b). Purification of LDs consisted in (i) treatment of cells to nutrient starvation (mostly nitrogen deprivation) to trigger LD formation, (ii) cell disruption, and (iii) LD isolation, generally through sucrose density centrifugation.…”

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“…This result is in line with the observations of Wong and Franz (2013) who found that Pt4 LDs ranged from 0.1 to 38 µm 3 in N-deficient conditions. Our results also tend to confirm what suggested Jaussaud et al (2020) and Leyland et al (2020) using epifluorescence microscopy. Indeed, Leyland et al (2020) observed a higher yield of LDs from cells disrupted by hypotonic shock in Pt4 compared to Pt1 with multiple small LDs for Pt1 and two relatively large LDs for Pt4.…”